Modification of reactivity of bone constructs

ABSTRACT

A method of enhancing the binding of growth factors and cell cultures to a demineralized allograft bone material which includes applying ex vivo an effective quantity of an ionic force change agent to at least a portion of the surface of a demineralized allograft bone material to produce a binding-sensitized demineralized allograft bone material and implanting the binding-sensitized demineralized allograft bone material into a host bone. The ionic force change agent may include at least one of enzymes, pressure, chemicals, heat, sheer force, oxygen plasma, supercritical nitrogen, supercritical carbon, supercritical water or a combination thereof. A molecule, a cell culture, or a combination thereof is administered to the binding-sensitized demineralized allograft bone material.

BACKGROUND

Numerous approaches are being employed to improve the bone generationand repair cycle (also referred to as the bone repair cascade). Suchissues are paramount in the treatment of all bone related defectsrelated to degeneration, injury, infection, malignancy or developmentalmalformation. For example, in the spinal surgery field, there areseveral different types of autologous bone graft substitutes that areeither currently available or are in various stages of development foruse in spine fusion surgery.

The use of bone grafts and bone substitute materials in orthopedicmedicine is known. While bone wounds can regenerate without theformation of scar tissue, fractures and other orthopedic injuries take along time to heal, during which time the bone is unable to supportphysiologic loading unaided. Metal pins, screws, rods, plates and meshesare frequently required to replace the mechanical functions of injuredbone. However, metal is significantly more stiff than bone. Use of metalimplants may result in decreased bone density around the implant sitedue to stress shielding. Physiologic stresses and corrosion may causemetal implants to fracture. Unlike bone, which can heal small damagecracks through remodeling to prevent more extensive damage and failure,damaged metal implants can only be replaced or removed. The naturalcellular healing and remodeling mechanisms of the body coordinateremoval of bone and bone grafts by osteoclast cells and formation ofbone by osteoblast cells.

Conventionally, bone tissue regeneration is achieved by filling a bonerepair site with a bone graft. Over time, the bone graft is incorporatedby the host and new bone remodels the bone graft. In order to place thebone graft, it is common to use a monolithic bone graft or to form anosteoimplant comprising particulated bone in a carrier. The carrier isthus chosen to be biocompatible, to be resorbable, and to have releasecharacteristics such that the bone graft is accessible. Generally, theformed implant, whether monolithic or particulated and in a carrier, issubstantially solid at the time of implantation and thus does notconform to the implant site. Further, the implant is substantiallycomplete at the time of implantation and thus provides little abilityfor customization, for example by the addition of autograft.

The use of bone grafts is generally limited by the available shape andsize of grafts. Bone grafts using cortical bone remodel slowly becauseof their limited porosity. Traditional bone substitute materials andbone chips are more quickly remodeled but cannot immediately providemechanical support. In addition, while bone substitute materials andbone chips can be used to fill oddly shaped bone defects, such materialsare not as well suited for wrapping or resurfacing bone.

Demineralized bone matrix (DBM) is demineralized allograft bone withosteoinductive activity. DBM is prepared by acid extraction of allograftbone, resulting in loss of most of the mineralized component butretention of collagen and noncollagenous proteins, including growthfactors. DBM does not contain osteoprogenitor cells, but the efficacy ofa demineralized bone matrix as a bone-graft substitute or extender maybe influenced by a number of factors, including the sterilizationprocess, the carrier, the total amount of bone morphogenetic protein(BMP) present, and the ratios of the different BMPs present. DBMincludes demineralized pieces of cortical bone to expose theosteoinductive proteins contained in the matrix. These activateddemineralized bone particles are usually added to a substrate or carrier(e.g. glycerol or a polymer). DBM is mostly an osteoinductive product,but lacks enough induction to be used on its own in challenging healingenvironments such as posterolateral spine fusion.

Allograft bone is a reasonable graft substitute for autologous bone. Itis readily available from cadavers and avoids the surgical complicationsand patient morbidity associated with harvesting autologous bone.Allograft bone is essentially a load-bearing matrix comprisingcross-linked collagen, hydroxyapatite, and osteoinductive bonemorphogenetic proteins. Human allograft tissue is widely used inorthopaedic surgery.

Even though allograft tissue has certain advantages over the othertreatments, one of the main drawbacks of the allograft treatment is thatthe ingrowth of the host bone into the grafted bone may take longer thanin an autograft. As a result, allograft treatment may be less effectivethan the autograft. Attempts have been made to overcome these drawbacksby modifying the bone graft's surface.

Despite the advances recently made in the art, new methods promotingingrowth of the host bone into the grafted bone are needed to betterutilize the advantages of allograft treatment.

SUMMARY

The present disclosure fills the foregoing need by providing devices(e.g., medical devices), systems and methods for enhancing ingrowth ofhost bone. In particular, the present disclosure provides anadvantageous allograft bone composition comprising modifyingdemineralized allograft bone to provide an ionic gradient to produce amodified demineralized allograft bone material, and implanting themodified demineralized allograft bone material. This aspect of thepresent disclosure may further provide administering to the modifieddemineralized allograft bone material a molecule, a cell culture or acombination thereof.

According to one aspect, a method for enhancing ingrowth of host bone isprovided comprising the steps of modifying at least a portion of ademineralized allograft bone material to provide an ionic gradient toproduce a modified demineralized allograft bone material; and implantingthe modified demineralized allograft bone material.

According to another aspect, a method of enhancing the binding of growthfactors and cell cultures to a demineralized allograft bone material isprovided comprising the steps of: applying ex vivo an effective quantityof an ionic force change agent to at least a portion of the surface of ademineralized allograft bone material to produce a binding-sensitizeddemineralized allograft bone material; implanting saidbinding-sensitized demineralized allograft bone material into a hostbone; and administering to said, binding-sensitized demineralizedallograft bone material a molecule, a cell culture, or a combinationthereof.

According to yet another aspect, a bone graft material is providedcomprising: a biocompatible material comprising a demineralizedallograft bone material, wherein at least a portion of a demineralizedallograft bone material is modified to provide an ionic gradient toproduce a binding-sensitized demineralized allograft bone material.

While multiple embodiments are disclosed, still other embodiments of thepresent disclosure will become apparent to those skilled in the art fromthe following detailed description, which is to be read in connectionwith the accompanying drawing(s). As will be apparent, the disclosure iscapable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present disclosure.Accordingly, the detailed description is to be regarded as illustrativein nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWING

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawing(s) where:

FIG. 1 is an exemplary block diagram of a method for enhancing thebinding of growth factors and cell cultures to a demineralized allograftbone material.

DEFINITIONS

To aid in the understanding of the disclosure, the followingnon-limiting definitions are provided:

“Bioactive agent or bioactive compound,” as used herein, refers to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. In some embodiments, the bioactive agent is a growthfactor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGD.

“Biocompatible,” as used herein, refers to materials that, uponadministration in vivo, do not induce undesirable long-term effects.

“Bone,” as used herein, refers to bone that is cortical, cancellous orcortico-cancellous of autogenous, allogenic, xenogenic, or transgenicorigin.

“Demineralized,” as used herein, refers to any material generated byremoving mineral material from tissue, e.g., bone tissue. In certainembodiments, the demineralized compositions described herein includepreparations containing less than 5% calcium and preferably less than 1%calcium by weight. Partially demineralized bone (e.g., preparations withgreater than 5% calcium by weight but containing less than 100% of theoriginal starting amount of calcium) is also considered within the scopeof the disclosure. In some embodiments, demineralized bone has less than95% of its original mineral content. Demineralized is intended toencompass such expressions as “substantially demineralized,” “partiallydemineralized,” and “fully demineralized.”

“Demineralized bone matrix,” as used herein, refers to any materialgenerated by removing mineral material from bone tissue. In someembodiments, the DBM compositions as used herein include preparationscontaining less than 5% calcium and preferably less than 1% calcium byweight. Partially demineralized bone (e.g., preparations with greaterthan 5% calcium by weight but containing less than 100% of the originalstarting amount of calcium) are also considered within the scope of thedisclosure.

“Osteoconductive,” as used herein, refers to the ability of anon-osteoinductive substance to serve as a suitable template orsubstance along which bone may grow.

“Osteogenic,” as used herein, refers to the ability of an agent,material, or implant to enhance or accelerate the growth of new bonetissue by one or more mechanisms such as osteogenesis, osteoconduction,and/or osteoinduction.

“Osteoimplant,” as used herein, refers to any bone-derived implantprepared in accordance with the embodiments of this disclosure andtherefore is intended to include expressions such as bone membrane, bonegraft, etc.

“Osteoinductive,” as used herein, refers to the quality of being able torecruit cells from the host that have the potential to stimulate newbone formation. Any material that can induce the formation of ectopicbone in the soft tissue of an animal is considered osteoinductive.

“Superficially demineralized,” as used herein, refers to bone-derivedelements possessing at least about 90 weight percent of their originalinorganic mineral content, the expression “partially demineralized” asused herein refers to bone-derived elements possessing from about 8 toabout 90 weight percent of their original inorganic mineral content andthe expression “fully demineralized” as used herein refers to bonecontaining less than 8% of its original mineral context.

The term “allograft” refers to a graft of tissue obtained from a donorof the same species as, but with a different genetic make-up from, therecipient, as a tissue transplant between two humans.

The term “autologous” refers to being derived or transferred from thesame individual's body, such as for example an autologous bone marrowtransplant.

The term “morbidity” refers to the frequency of the appearance ofcomplications following a surgical procedure or other treatment.

The term “osteoinduction” refers to the ability to stimulate theproliferation and differentiation of pluripotent mesenchymal stem cells(MSCs). In endochondral bone formation, stem cells differentiate intochondroblasts and chondrocytes, laying down a cartilaginous ECM, whichsubsequently calcifies and is remodeled into lamellar bone. Inintramembranous bone formation, the stem cells differentiate directlyinto osteoblasts, which form bone through direct mechanisms.Osteoinduction can be stimulated by osteogenic growth factors, althoughsome ECM proteins can also drive progenitor cells toward the osteogenicphenotype.

The term “osteoconduction” refers to the ability to stimulate theattachment, migration, and distribution of vascular and osteogenic cellswithin the graft material. The physical characteristics that affect thegraft's osteoconductive activity include porosity, pore size, andthree-dimensional architecture. In addition, direct biochemicalinteractions between matrix proteins and cell surface receptors play amajor role in the host's response to the graft material.

The term “osteogenic” refers to the ability of a graft material toproduce bone independently. To have direct osteogenic activity, thegraft must contain cellular components that directly induce boneformation. For example, a collagen matrix seeded with activated MSCswould have the potential to induce bone formation directly, withoutrecruitment and activation of host MSC populations. Because manyosteoconductive scaffolds also have the ability to bind and deliverbioactive molecules, their osteoinductive potential will be greatlyenhanced.

The term “patient” refers to a biological system to which a treatmentcan be administered. A biological system can include, for example, anindividual cell, a set of cells (e.g., a cell culture), an organ, or atissue. Additionally, the term “patient” can refer to animals,including, without limitation, humans.

The term “treating” or “treatment” of a disease refers to executing aprotocol, which may include administering one or more drugs to a patient(human or otherwise), in an effort to alleviate signs or symptoms of thedisease. Alleviation can occur prior to signs or symptoms of the diseaseappearing, as well as after their appearance. Thus, “treating” or“treatment” includes “preventing” or “prevention” of disease. Inaddition, “treating” or “treatment” does not require completealleviation of signs or symptoms, does not require a cure, andspecifically includes protocols, which have only a marginal effect onthe patient.

The term “xenograft” refers to tissue or organs from an individual ofone species transplanted into or grafted onto an organism of anotherspecies, genus, or family.

DETAILED DESCRIPTION

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “1 to 10” includes any and allsubranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all subranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

Aspects of the present disclosure provide reagents, methods and systemsfor enhancing ingrowth of host bone. Applicants have found thatmodifying a demineralized allograft bone material to provide an ionicgradient to produce a modified demineralized allograft bone material,and implanting the modified demineralized allograft bone materialresults in enhanced ingrowth of host bone.

In one embodiment of the present disclosure an ionic force change agentis applied to modify the demineralized allograft bone material.According to one embodiment of the disclosure, the demineralizedallograft bone material may comprise a demineralized bone matrix (DBM)comprising fibers, particles and any combination of thereof. Accordingto another embodiment, a bone graft structure may be used whichcomprises a composite bone which includes a bone powder, a polymer and ademineralized bone.

The ionic force change agent may be a binding agent, which modifies thedemineralized allograft bone material or bone graft structure to bindmolecules, such as, for example, growth factors, or cells, such as, forexample, cultured cells, or a combination of molecules and cells. In thepractice of the disclosure the growth factors include but are notlimited to BMP-2, rhBMP-2, BMP-4, rhBMP-4, BMP-6, rhBMP-6, BMP-7 (OP-1),rhBMP-7, GDF-5, LIM mineralization protein, platelet derived growthfactor (PDGF), transforming growth factor-β (TGF-β), insulin-relatedgrowth factor-I (IGF-I), insulin-related growth factor-II (IGF-II),fibroblast growth factor (FGF), beta-2-microglobulin (BDGF II), andrhGDF-5. A person of ordinary skill in the art will appreciate that thedisclosure is not limited to growth factors only. Other molecules canalso be employed in the disclosure. For example, tartrate-resistant acidphosphatase, which is not a growth factor, may also be used in thedisclosure.

If a cell culture is employed, the cells include but are not limited tomesenchymal stems cells, pluripotent stem cells, embryonic stem cells,osteoprogenitor cells, osteoblasts, osteoclasts, and any bonemarrow-derived cell lines.

In one embodiment of the method of the disclosure, the ionic forces ofthe demineralized allograft bone material are changed by a one-to-onesubstitution of the calcium ion with an element selected from the groupconsisting of lithium, sodium, potassium and cesium ions ofhydroxyapatite.

Another aspect of the present disclosure provides a method of enhancingthe binding of molecules and cell cultures to a demineralized allograftbone material comprising applying ex vivo an effective quantity of anionic force change agent to the demineralized allograft bone material toproduce a binding-sensitized demineralized allograft bone material; andimplanting said binding-sensitized demineralized allograft bone materialinto a host bone. It may be desirable to administer to thebinding-sensitized demineralized allograft bone material a molecule, acell culture or a combination thereof all of which are capable ofbinding to said binding-sensitized demineralized allograft bonematerial. For example, the molecule may be a growth factor such as, forexample, BMP-2, rhBMP-2, BMP-4, rhBMP-4, BMP-6, rhBMP-6, BMP-7 (OP-1),rhBMP-7, GDF-5, LIM mineralization protein, platelet derived growthfactor (PDGF), transforming growth factor β (TGF-β), insulin-relatedgrowth factor-I (IGF-I), insulin-related growth factor-II (IGF-II),fibroblast growth factor (FGF), beta-2-microglobulin (BDGF II), andrhGDF-5. Other molecules can also be employed in the disclosure, suchas, for example, tartrate-resistant acid phosphatase, which is not agrowth factor.

Cells may also be used instead of or in addition to molecules, such asgrowth factors. Non-limiting examples of suitable cell types includemesenchymal stems cells, pluripotent stem cells, embryonic stem cells,osteoprogenitor cells, osteoblasts and osteoclasts.

Depending upon the condition of the patient, new bone ingrowth isaccomplished by one or more mechanisms such as osteogenesis,osteoconduction and osteoinduction. It can be appreciated that the needsof a child are different from an aging patient afflicted withosteoporosis. Accordingly, there is no “one size fits all” approachtowards optimizing the healing conditions in a patient.

In one aspect, the disclosure relates to a method of modifying ademineralized allograft bone material (also referred to as an implant)in such a way that the original chemical forces naturally present havebeen altered in such a way as to attract and bind growth factors, otherproteins and cells affecting osteogenesis, osteoconduction andosteoinduction.

Intramolecular or intermolecular attractions between atoms are formedthrough weak chemical forces, which include hydrogen bonds, van derWaals forces, ionic bonds and hydrophobic interactions. These weakforces create bonds that are constantly forming and breaking atphysiological temperature and are readily reversible under physiologicalconditions. The transient bonds between metabolites and macromolecules,and hormones and receptors, and all the other cellular moietiesnecessary for life are required for biomolecular interactions sincerigid, static bonds will inhibit, if not paralyze, cellular activities.

In one aspect of the disclosure, the implant or demineralized allograftbone material is modified in such a way that the original chemicalforces naturally present are altered so that the implant attracts andbinds proteins, such as, for example, growth factors and cells,including cells from cell cultures.

The disclosure provides for a method of enhancing ingrowth of host boneby modifying a bone graft material, in particular demineralizedallograft bone material, to provide a gradient, implanting the modifieddemineralized allograft bone material, and administering to the modifieddemineralized allograft bone material a molecule, such as, for example,a growth factor, and/or a cell culture. A person of ordinary skill inthe art will appreciate that the molecule and/or the cell culture may beadministered to the modified demineralized allograft bone materialbefore and/or after implanting the modified demineralized allograft bonematerial into the host bone.

Non-limiting examples of a bone graft material include demineralizedbone matrix, or a bone composite.

According to some embodiments of the disclosure, the demineralized bonematrix may comprise demineralized bone matrix fibers and/ordemineralized bone matrix chips. In some embodiments, the demineralizedbone matrix may comprise demineralized bone matrix fibers anddemineralized bone matrix chips in a 30:60 ratio.

According to one embodiment of the disclosure, the bone compositecomprises a bone powder, a polymer and a demineralized bone. Indifferent embodiments of the disclosure, bone powder content can rangefrom about 5% to about 90% w/w, polymer content can range from about 5%to about 90% w/w, and demineralized bone particles content comprises thereminder of the composition. Preferably, the demineralized boneparticles comprise from about 20% to about 40% w/w while the polymer andthe bone powder comprise each from about 20% to about 60% w/w of thecomposition. The bone graft materials of the present disclosure includethose structures that have been modified in such a way that the originalchemical forces naturally present have been altered to attract and bindmolecules, including, without limitation, growth factors and/or cells,including cultured cells.

The disclosure also discloses a method of enhancing binding ofmolecules, such as, for example, growth factors and cell cultures byapplying ex vivo an effective quantity of an ionic force change agent toa demineralized allograft bone material, in particular, the surface of ademineralized allograft bone material, to produce a binding-sensitizeddemineralized allograft bone material, and implanting thebinding-sensitized demineralized allograft bone material. An effectiveamount of molecules, including growth factors and cell cultures can beadministered to the binding-sensitized demineralized allograft bonematerial both before and after implanting the modified demineralizedallograft bone material into the host bone.

In another aspect, the disclosure involves the addition of an ionicforce change agent to the demineralized allograft bone material therebymodifying its surface, namely modifying its charge in a targeted mannerto produce an appropriately charged demineralized allograft bonematerial. The ionic force change agent may be applied to the entiredemineralized allograft bone material or to selected portions/surfacesthereof.

In some embodiments, the ionic force change agent comprises at least oneof enzymes, enzyme mixtures, pressure (e.g., isostatic pressure),chemicals, heat, sheer force, oxygen plasma, or a combination thereof.For example, the ionic force change agent may comprise an enzyme such ascollagenase or pepsin, which can be administered for a sufficient periodof time to partially digest at least a portion of the demineralizedallograft bone material. Subsequently, the enzyme may be deactivatedand/or removed.

Any enzyme or enzyme mixture may be contemplated, and treatment timedurations may be altered in accordance with the enzyme(s) used. Somesuitable enzymes that may degrade the DBM material include, but are notlimited to, cysteine proteinases, matrix metalloproteinases, enzymessuch as amylases, proteases, lipases, pectinases, cellulases,hemicellulases, pentosanases, xylanases, phytases or combinationsthereof. Exemplary enzymes suitable to partially degrade and modify theDBM material, include but are not limited to, cathepsin L, cathepsin K,cathepsin B, pepsin, plasminogen, elastase, stromelysin, plasminogenactivators, matrix metalloproteinases (MMPs), including but not limitedto collagenase and gelatinase, or a combination thereof. In someembodiments, the DBM material can be subjected to pressure to modify it.The simplest pressing technique is to apply pressure to theunconstrained DBM material. Examples include pressing the DBM materialusing a mortar and pestle, applying a rolling/pressing motion such as isgenerated by a rolling pin, or pressing the bone pieces between flat orcurved plates. These flattening pressures cause the DBM material fibersto remain intact.

Another pressing technique involves mechanically pressing demineralizedbone material, which can be constrained within a sealed chamber having ahole (or a small number of holes) in its floor or bottom plate. Theseparated fibers extrude through the holes with the hole diameterlimiting the maximum diameter of the extruded fibers. This constrainedtechnique results in fibers that are largely intact (as far as length isconcerned).

In a combined unconstrained/constrained pressing technique that resultsin longer fibers by minimizing fiber breakage, the demineralized bone isfirst pressed into an initially separated mass of fibers while in theunconstrained condition and thereafter these fibers are constrainedwithin the sealed chamber where pressing is continued.

In general, pressing of demineralized bone to provide demineralized boneparticles can be carried out at from about 1,000 to about 40,000 psi,and preferably at from about 5,000 to about 20,000 psi.

Subsequent to the addition of the ionic force change agent, thepractitioner may optionally administer an appropriate molecule or cellculture. Generally, the molecule or cell culture is applied withinminutes, for example from about 1 to about 120 minutes beforeimplantation into the patient.

One class of molecules suitable for one embodiment of the disclosure isgrowth factors. Growth factors suitable for use in the practice of thedisclosure include but are not limited to bone morphogenic proteins, forexample, BMP-2, rhBMP-2, BMP-4, rhBMP-4, BMP-6, rhBMP-6, BMP-7 (OP-1),rhBMP-7, GDF-5, and rhGDF-5. Bone morphogenic proteins have been shownto be excellent at growing bone and there are several products beingtested. For example, rhBMP-2 delivered on an absorbable collagen sponge(INFUSE° Bone Graft, Medtronic Sofamor Danek, Memphis, Term.) has beenused inside titanium fusion cages and resulted in successful fusion andcan be used on a ceramic carrier to enhance bone growth in aposterolateral fusion procedure. rhBMP-2 can also be used on a carrierfor acute, open fractures of the tibial shaft. BMP-7 (OP-1) alsoenhances bone growth in a posterolateral fusion procedure.

Additionally, suitable growth factors include, without limitation, LIMmineralization protein, platelet derived growth factor (PDGF),transforming growth factor β (TGF-β), insulin-related growth factor-I(IGF-I), insulin-related growth factor-II (IGF-II), fibroblast growthfactor (FGF), and beta-2-microglobulin (BDGF II).

Further, molecules, which do not have growth factor properties may alsobe suitable for this disclosure. An example of such molecules istartrate-resistant acid phosphatase.

In one embodiment, the demineralized allograft bone material is treatedwith a negatively-charged ionic force change agent to produce anegatively-charged demineralized allograft bone material. Thenegatively-charged demineralized allograft bone material attracts apositively charged molecule having a pI from about 8 to about 10.Examples of positively charged molecules having a pI from about 8 toabout 10 include but are not limited to, rhBMP-2 and rhBMP-6.

In another embodiment, the demineralized allograft bone material istreated with a positively-charged ionic force change agent such that thepositively-charged demineralized allograft bone material attracts amolecule with a slightly negative charge, for example a charge of pIabout 5 to about 7. Examples of molecules having a slightly negativecharge include rhBMP-4.

In yet another embodiment, the demineralized allograft bone material istreated with a positively-charged ionic force change agent to produce apositively-charged demineralized allograft bone material such thatcells, in particular cell cultures having a negative surface charge bindto the positively-charged demineralized allograft bone material.Examples of cells which are suitable for use in the practice of thedisclosure include but are not limited to mesenchymal stems cells,pluripotent stem cells, embryonic stem cells, osteoprogenitor cells andosteoblasts.

The mechanisms by which a demineralized allograft bone material mayacquire ionic forces include but are not limited to ionization, ionadsorption and ion dissolution.

In one embodiment, the implant is modified to give it the selectedcharge by a one-to-one substitution of the calcium ion with lithium,sodium, potassium or cesium of hydroxyapatite.

In yet another aspect, treatments with gradient-affecting elements, suchas elements present in hydroxyapatite, and human proteins are employed.Suitable gradient-affecting proteins are those present in the organicphase of human bone tissue. The gradient-affecting proteins derivemolecule or cell attraction without the potential damaging effects onthe implants, as may be the case with other chemical treatments. Usuallythis is accomplished through surface treatments such as, for example,plasma treatment to apply an electrostatic charge on bone.

The term “plasma” in this context is an ionized gas containing excitedspecies such as ions, radicals, electrons and photons. The term “plasmatreatment” refers to a protocol in which a surface is modified using aplasma generated from process gases including, but not limited to, O₂,He, N₂, Ar and N₂O. To excite the plasma, energy is applied to thesystem through electrodes. This power may be alternating current (AC),direct current (DC), radiofrequency (RF), or microwave frequency (MW).The plasma may be generated in a vacuum or at atmospheric pressure. Theplasma can also be used to deposit polymeric, ceramic or metallic thinfilms onto surfaces. Plasma treatment is an effective method touniformly alter the surface properties of substrates having different orunique size, shape and geometry including but not limited to bone andbone composite materials.

FIG. 1 is an exemplary block diagram of a method for enhancing thebinding of growth factors and cell cultures to a demineralized allograftbone material. In step 101, an ionic force change agent is applied exvivo to at least a portion of a surface of a demineralized allograftbone material (e.g., demineralized bone matrix (DBM) material fibers orDBM material particles) to produce a binding-sensitized demineralizedallograft bone material. In some embodiments, the ionic force changeagent may comprise at least one of enzymes, pressure (e.g., isostaticpressure), chemicals, heat, sheer force or plasma to alter and/or chargethe surface, to render the demineralized bone matrix material morereactive, chemically and/or physically.

For example, such surface treatment or modification of demineralizedbone matrix material advantageously exposes functional groups so as toincrease reactivity and bonding with other molecules. In particular,this would enhance reactivity of the demineralized bone matrix materialso that it can better immobilize or bind with drugs, proteins, or othermolecules when, e.g., implanted in vivo.

In some embodiments, the ionic force change agent may comprise anenzyme, such as, e.g., collagenase or pepsin. Usage of alternativeenzymes may be contemplated. The demineralized bone matrix material maybe treated with an enzyme or enzyme mixture for a period of time, e.g.,sufficient to cause at least a portion of the demineralized bone matrixmaterial to become at least partially digested. Subsequently, theenzyme/enzyme mixture may be deactivated. This enzyme treatment willcause the demineralized bone matrix material to become “sticky” and thuscause physical entanglement of the demineralized bone matrixparticles/fibers and/or improve reactivity of the demineralized bonematrix particles/fibers with other molecules.

In optional step 103, a molecule, a cell culture or any combinationthereof, may be administered to the binding-sensitized allograft bonematerial prior to implantation. For example, drugs, proteins or othermolecules which are desired to be bound to the DBM material may beadministered to the binding-sensitized demineralized allograft bonematerial.

In step 105, the binding-sensitized demineralized allograft bonematerial (with or without additional molecules and/or cell culturesbound thereto) may be implanted into a host bone. In step 107, amolecule, a cell culture or any combination thereof, may be administeredto the binding-sensitized allograft bone material after implantation.

A modified demineralized allograft bone material may be used inconjunction with a delivery system for delivering the modifieddemineralized allograft bone material to a surgical site. The deliverysystem may comprise a covering wherein the modified demineralizedallograft bone material is provided within the covering for delivery tothe surgical site. Generally, the covering may be a single ormulti-compartment structure capable of at least partially retaining themodified demineralized allograft bone material provided therein untilthe covering is placed at a surgical site. Upon placement, the coveringfacilitates transfer of the modified demineralized allograft bonematerial surrounding the surgical site. The covering may participate in,control, or otherwise adjust, the release of the substance orpenetration of the covering by surrounding materials, such as cells ortissues.

A covering according to some embodiments may comprise natural andsynthetic polymers which provide and extended and/or increased shelflife to the delivery system. The extended shelf life of the polymersprevents environmental degradation of the covering, thus increasingefficiency, preventing waste and providing a more effective, usableproduct over an extended period of time.

In some embodiments, the covering may be used for containment ofparticulate or morselized materials (the substance provided in thecovering), optionally to provide a focus or concentration of biologicalactivity. In some embodiments, the covering may be used for maintainingmaterials (the substance provided in the covering) in spatial proximityto one another, possibly to provide a synergistic effect. In someembodiments, the delivery system may be used to control availability ofsubstances provided within the delivery system to cells and tissues of asurgical site over time. In some embodiments, the delivery system may beused for delivery through a limited opening, such as in minimallyinvasive surgery or mini-open access. In some embodiments, the deliverysystem may be used to deliver morselized or particulated materials (thesubstance provided in the covering) in pre-measured amounts. In otherembodiments, the substance may be liquid or flowable, or combinations ofthese with particulate, morselized, and/or other materials.

In various embodiments, the covering can contain a demineralizedallograft material. The covering limits, and in some embodimentseliminates graft migration and maintains graft density. The deliverysystem, with contained demineralized allograft material, may beconfigured to conform to surrounding bony contours or implant space. Insome embodiments, the delivery system provides a pathway forhealing/cell penetration and tissue ingrowth. Thus, the covering mayfacilitate transfer of a substance out of the covering or transfer orsurrounding materials at the surgical site, such as cells and tissues,into the covering.

The covering may have a single compartment or may have a plurality ofcompartments. Thus, in one embodiment, the covering is dual-compartmentand comprises first and second compartments. A first substance may beprovided in the first compartment and a second substance may be providedin the second compartment. The second compartment may be adjacent to,apart from, inside, or surrounding the first compartment. Materialsforming the first compartment and the second compartment may be the sameor different. Selection of materials, positioning of the compartments,and other factors relating to the first and second compartments may bechosen to achieve simultaneous or sequential delivery or release of asubstance or substances.

Covering Material

The covering may comprise a structural material and, in someembodiments, a functional material. The structural material may comprisea mesh material, a polymeric material, or other. The functional materialmay comprise, for example, a radiopaque material, a bacteriocidalmaterial, or other material.

In various embodiments, in accordance with the specific application forwhich the covering is being used, the covering may be rigid, may beflexible, may be non-elastic, or may be elastic. The covering materialmay be braided, woven, non-woven shape memory, particulate, threaded,porous, or non-porous.

The covering may participate in, control, or otherwise adjust therelease of the substance. For example, the covering may act as aselectively permeable membrane and/or may be porous, with the level ofporosity being related to the nature of the substances inside thecovering. Thus, the material for and configuration of the covering maybe selected or adjusted based on desired release characteristics.Specific properties that may be adjusted include thickness,permeability, porosity, strength, flexibility, elasticity, and others ofthe covering material. It is to be appreciated that some of theseproperties may depend on others. For example, the thickness and porosityof the material may contribute to its strength, flexibility, andelasticity.

In some embodiments, the covering may be porous to fluid and/or cells,may be biocompatible, and may be resistant to rupture (including shouldthe substance provided therein swell). In some embodiments, the coveringwith the demineralized allograft material provided therein may beloadbearing. The covering may be resorbable or non-resorbable. Thecovering may provide increased handling properties, may have irrigationresistance, and/or may support cellular penetration. Flexibility of thecovering may be selected to suit particular applications. In someapplications, it may be desirable to have a flexible covering.

If the covering is made from a resorbable material, the coveringdegrades and disappears after a period of time. If the covering is notmade of a resorbable material, the covering remains in the body. Tissueingrowth may occur to bind the host tissue to the substance providedwithin the covering. Tissue ingrowth through and around the covering,between the host tissue and the substance provided within the covering,may be promoted via openings in the covering.

In various embodiments, the covering may comprise a porous material or amesh material. The size of the pores of the covering may be designed topermit cellular infiltration (approximately several microns to severalmillimeters), but may also be designed specifically to exclude cells forthe inside of the covering (e.g. approximately 0.45 microns) and onlyallow diffusion of small molecules (proteins and hormones). Thus, thecovering may act to control access to the interior of the deliverysystem by cells. In embodiments comprising more than one compartment,characteristics of the covering material may be varied betweencompartments. Generally, the porosity, flexibility, strength, or anyother characteristic of one compartment may vary from thatcharacteristic of the other compartment.

The covering may be formed of a resorbable or nonresorbable, natural orsynthetic biocompatible material. In some embodiments, more than onematerial may be used, including as multiple layers. For example, in anembodiment comprising two compartments, one or more materials may beused for the first compartment and a different material or materials maybe used for the second compartment. For example, one compartment orportions thereof may be made of material or materials that provide adesired property or properties relative to other compartments orportions thereof, such as increased or decreased resorbability orstiffness, or the different compartments or portions thereof may beimparted with different drug delivery properties, etc. Alternatively,all compartments may comprise the same material or mixtures ofmaterials. Where the characteristics of the material are varied betweencompartments, or over the surface of a single compartment, the pores ofthe first compartment or portion thereof may be larger than the pores ofthe second compartment.

The covering may comprise any suitable structure for delivering asubstance in vivo. Thus, as described, the covering may comprise a mesh.In other embodiments, the covering may comprise a polymeric structurewith a chamber provided therein. The chamber may be filled with asubstance for delivering in vivo, such as demineralized allograftmaterial, fully mineralized bone material, or others disclosed herein.

In some embodiments, the covering may expand when placed in the body.Expansion can be provided in at least two ways: the covering may becompressed such that the covering expands when placed in the body or thecovering may be made of a material that expands when it comes in contactwith water or other bodily fluids, either by way of liquid absorption,or by stretching when the materials inside it absorb liquid andthemselves expand. In some embodiments, the covering may comprise ashape memory material such as copper-zinc-aluminum-nickel alloy,copper-aluminum-nickel alloy, and nickel-titanium (NiTi) alloy.Reinforcing materials such as cortical bone, calcium phosphates, etc.may be incorporated into the structure of the covering to reinforce it.

The covering may be configured for specific compressive strength andrigidity by adjusting density and resorption time of the covering. Insome embodiments, a coating may be provided over the covering. Forexample, the coating may be a compound of poly-L-lactide, ofpolyglycolic acid, or their polymers. The coating may be selected suchthat it has a resorption time wherein it is resorbed by the body and thematerial within the covering is permitted to exit through openings inthe covering.

Exemplary Covering Materials

A covering according to an aspect of the present disclosure, thecovering may comprise demineralized allograft material and at least oneof bioerodible polymers, bioabsorbable polymers, biodegradablebiopolymers, synthetic polymers, copolymers and copolymer blends andcombinations thereof. Exemplary materials may include biopolymers andsynthetic polymers such as human skin, human hair, bone sheets,collagen, fat, thin cross-linked sheets containing fibers and/or fibersand chips, degradable sheets made from polyethylene glycol (PEG),chitosan sheets, alginate sheets, cellulose sheets, hyaluronic acidsheet, as well as copolymer blends of poly (lactide-co-glycolide) PLGA.

Exemplary materials may include polymeric material, woven material andbraided material, non-woven; shape memory material; using outerparticles to contain inner particles; attach particles to threads; addporosity to mesh fibers; non-porous materials; non-porous materials. Insome embodiments, materials may be used for portions of the covering,such as for a compartment of the covering, that are substantiallyimpenetrable.

In some embodiments, the covering may comprise a mesh material. Suitablemesh materials include natural materials, synthetic polymeric resorbablematerials, synthetic polymeric non-resorbable materials, and othermaterials. Natural mesh materials include silk, extracellular matrix(such as DBM, collagen, ligament, tendon tissue, or other),silk-elastin, elastin, collagen, and cellulose. Synthetic polymericresorbable materials include poly(lactic acid) (PLA), poly(glycolicacid) (PGA), poly(lactic acid-glycolic acid) (PLGA), polydioxanone, PVA,polyurethanes, polycarbonates, and others. Other suitable materialsinclude carbon fiber, metal fiber, and various meshes. In otherembodiments, the covering may comprise non-woven material such as spuncocoon or shape memory materials having a coil shape or shape memoryalloys.

Generally, the covering may be formed of any natural or syntheticstructure (tissue, protein, carbohydrate) that can be used to form acovering configuration. Thus, the covering may be formed of a polymer(such as polyalkylenes (e.g., polyethylenes, polypropylenes, etc.),polyamides, polyesters, poly(glaxanone), poly(orthoesters),poly(pyrolicacid), poly(phosphazenes), polycarbonate, otherbioabsorbable polymer such as Dacron or other known surgical plastics, anatural biologically derived material such as collagen, gelatin,chitosan, alginate, a ceramic (with bone-growth enhancers,hydroxyapatite, etc.), PEEK (polyether-etherketone), dessicatedbiodegradable material, metal, composite materials, a biocompatibletextile (e.g., cotton, silk, linen), extracellular matrix components,tissues, or composites of synthetic and natural materials, or other.Various collagen materials can be used, alone or in combination withother materials, including collagen sutures and threads. Any suitablecollagen material may be used, including known collagen materials. Someexamples include polymer or collagen threads woven, or knitted into amesh. Other suitable materials include thin polymer sheets molded in thepresence of a porogen and having underwent leaching; polymer sheets ornaturally derived sheets such as fascia and other collagen materials,small intestinal submucosa, or urinary bladder epithelium, the sheetsbeing punctured to introduce porosity; specific shapes printed usingavailable or future printing technologies; naturally secreted materialssuch as bacterial cellulose grown within specific molds; etc.

In some embodiments, mesh fibers may be treated to impart porosity tothe demineralized allograft material that is in fiber form. This may bedone, for example, to PLA, PLGA, PGA, and other fibers. One suitablemethod for treating the mesh fibers comprises supercritical carbondioxide, supercritical nitrogen, or supercritical water treatment topartially solubilize the particles. This treatment may further becarried out for viral inactivation. Another suitable method for treatingthe mesh fibers comprises explosive decompression. Explosivedecompression generates porosity and leads to controlled permeability.The mesh material further may be loaded with cells, growth factors, orbioactive agents.

In further embodiments, fibers of a mesh material may be treated such asby having particles adhered thereto. The particles may be, for example,bone particles, demineralized allograft material, or the like. Thus, inone embodiment, the covering may comprise a plurality of threads formedinto a fabric. The threads may have particles adhered thereto. Forexample, the threads may have particles strung on the thread. In analternative embodiment, the covering may be formed of a material and thematerial may be coated with particles.

In yet other embodiments, the covering may comprise a non-porousmaterial, which may be permeable. A non-porous material may be used forlater (or delayed) delivery of a substance provided therein. Suchsubstance may comprise, for example, cells, growth factors, or bonemorphogenetic proteins. Accordingly, in one embodiment, a deliverysystem for delayed delivery of cells, growth factors, or bonemorphogenetic proteins is provided comprising a non-porous covering.

In particular, in various embodiments, the device may comprise abioerodible, a bioabsorbable, and/or a biodegradable biopolymer that mayprovide immediate release, or sustained release of the clonidine.Examples of suitable sustained release biopolymers include but are notlimited to poly (alpha-hydroxy acids), poly (lactide-co-glycolide)(PLGA), polylactide (PLA), polyglycolide (PG), polyethylene glycol (PEG)conjugates of poly (alpha-hydroxy acids), poly(orthoester)s (POE),polyaspirins, polyphosphagenes, collagen, starch, pre-gelatinizedstarch, hyaluronic acid, chitosans, gelatin, alginates, albumin, fibrin,vitamin E compounds, such as alpha tocopheryl acetate, d-alphatocopheryl succinate, D,L-lactide, or L-lactide, -caprolactone,dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA,PEGT-PBT copolymer (polyactive), PEO-PPO-PAA copolymers, PLGA-PEO-PLGA,PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymers, SAIB(sucrose acetate isobutyrate) or combinations thereof. As persons ofordinary skill are aware, mPEG and/or PEG may be used as a plasticizerfor PLGA, but other polymers/excipients may be used to achieve the sameeffect. mPEG imparts malleability to the resulting formulations. In someembodiments, these biopolymers may also be coated on a medical device toprovide the desired release profile. In some embodiments, the coatingthickness may be thin, for example, from about 5, 10, 15, 20, 25, 30,35, 40, 45 or 50 microns to thicker coatings 60, 65, 70, 75, 80, 85, 90,95, 100 microns to delay release of the substance from the medicaldevice. In some embodiments, the range of the coating on the medicaldevice ranges from about 5 microns to about 250 microns or 5 microns toabout 200 microns to delay release from the medical device.

In various embodiments, the medical device comprisespoly(lactide-co-glycolide) (PLGA), polylactide (PLA), polyglycolide(PGA), D-lactide, D,L-lactide, L-lactide, D,L-lactide-co-ε-caprolactone,D,L-lactide-co-glycolide-co-ε-caprolactone, L-lactide-co-ε-caprolactoneor a combination thereof.

Functional Material Characteristics

The covering material may have functional characteristics.Alternatively, other materials having functional characteristics may beincorporated into the covering. Functional characteristics may includeradiopacity, bacteriocidity, source for released materials, tackiness,etc. Such characteristics may be imparted substantially throughout thecovering or at only certain positions or portions of the covering.

Suitable radiopaque materials include, for example, ceramics,mineralized bone, ceramics/calcium phosphates/calcium sulfates, metalparticles, fibers, and iodinated polymer. Polymeric materials may beused to form the covering and be made radiopaque by iodinating them.Other techniques for incorporating a biocompatible metal or metal saltinto a polymer to increase radiopacity of the polymer may also be used.Suitable bacteriocidal materials may include, for example, tracemetallic elements. In some embodiments, trace metallic elements may alsoencourage bone growth.

Functional material, such as radiopaque markers, may be provided at oneor more locations on the covering or may be provided substantiallythroughout the covering. Thus, for example, in a tubular covering, aradiopaque marker may be provided at a tip of the tubular covering. Suchmarker may facilitate placement of the covering. Radiopaque materialsmay be incorporated into the covering and/or into the substance fordelivery by the covering. Further, radiopaque materials may be providedat only some locations on the covering such that visualization of thoselocations provides indication of the orientation of the covering invivo.

The covering itself may be designed to release materials duringdegradation of the covering material. Thus, bone morphogenic proteins(BMPs), growth factors, antibiotics, angiogenesis promoting materials(discussed more fully below), bioactive agents (discussed more fullybelow), or other actively releasing materials may be incorporated intothe covering material such that as the covering material is degraded inthe body, the actively releasing material is released. For example, anactively releasing material may be incorporated into a biodegradablepolymer covering such as one manufactured of a biodegradable polyestersuch as poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(lactic-co-glycolic acid) (PLGA), or polyhydroxyalkanoates(polyhydroxybutyrates and polyhydroxyvalerates and copolymers). In someembodiments, poly(ethylene glycol) (PEG) may be incorporated into thebiodegradable polyester to add hydrophilic and other physico-chemicalproperties to enhance drug delivery. In some embodiments, composites ofallograft bone and biodegradable polymers (for example, PLEXUR® productsavailable from Osteotech™) may be used in the covering.

In some embodiments, the covering may comprise a material that becomestacky upon wetting. Such material may be, for example, a protein orgelatin based material. Tissue adhesives, including mussel adhesiveproteins and cyanoacrylates, may be used to impart tackiness to thecovering. In further examples, alginate or chitosan material may be usedto impart tackiness to the covering. In further embodiments, an adhesivesubstance or material may be placed on a portion of the covering or in aparticular region of the covering to anchor that portion or region ofthe covering in place at an implant site.

In one embodiment, the covering comprises two compartments, first andsecond materials may be used for the first and second compartments,respectively. The first material may release or expose a growth factoraccording to a first rate and the second material may release a growthfactor according to a second rate. Further, the growth factors releasedby the first and second compartments may be the same or may bedifferent. For example, an angiogenic growth factor may be provided withthe first compartment and an osteoinductive growth factor may beprovided with the second compartment in addition to the modifieddemineralized allograft material.

Mesh Formulation

Any suitable technique may be used for forming a material for thecovering. Generally, the material may be formed as a substantially solidmaterial, as a sheet, as a mesh, or in other configuration. In someembodiments, the material may be a textile type material. Thus, forexample, the material may be formed using a textile approach such as beweaving, rug making, knitting, etc. Such formation may be by amechanical or industrial method. In another embodiment, a substantiallysolid sheet may be formed and may be treated to assume a configurationpenetrable by cells, fluids, and proteins. For example, the sheet may beperforated, may be expanded to create openings, or other. Also, it wouldbe perfectly suitable to take a thin sheet of the covering material, andto perforate it, expand it to create openings, or otherwise make itpenetrable by cells, fluids and proteins.

In one embodiment, elongated bone-derived particles or fragments ofsmall intestinal submucosa may be combined longitudinally into threesmall bundles, each having, for example, from about 1 to about 3 tissueparticles. The three bundles may then be braided. Various methods ofbraiding and types of braids any of which may be useful in producing thematerial of the disclosure herein are also described. The ends of thebraided tissue-derived particles may then be glued together using afixation agent to prevent their unraveling, or they may be held togetherwith a biocompatible polymer or metal band.

In an alternative embodiment, the modified demineralized allograftmaterial may be combined with a solvent to form a material. Exemplarysolvents include water, lower alkanols, ketones, and ethers and mixturesof any of these or other materials. The material may then be extruded atan appropriate temperature and pressure to create a thread. Threads mayalso be produced by spinning, drawing, rolling, solvent-extruding,cutting or laser cutting from a sheet or bar stock. The material mayalternatively be cast or molded into a solid sheet or bar stock and thencut into thin threads. These may be used immediately or woven into amesh. Alternatively or in addition, they may be spliced, wrapped, plied,cabled, braided, woven, or some combination of these. The material maybe shaped by thermal or chemical bonding, or both. In one embodiment, aportion of the solvent is removed from the material before extrusion.

Alternatively or in addition, the material may be cast as a slurry,extruded, or molded and then mixed with the modified demineralizedallograft material. For example, the material may be solvent cast usinga press such as a Carver press to spread the material into a film.Solvent evaporation will yield a porous film. Alternatively, thematerial may be compression molded into a film. The mesh size orporosity of the film will depend on the thickness of the film and theviscosity of the precursor and can be easily manipulated by one skilledin the art. Where elongated particles are used in an extruded aggregate,they will tend to be aligned roughly parallel to one another.

In an alternative embodiment, a thread of a biocompatible natural orsynthetic material, for example, polylactide or collagen, may be coatedwith tissue-derived or other elements, for example, by dubbing. Forexample, a polymer fiber may be coated with an adhesive, for example,lecithin, and bone particles or other osteoconductive or osteoinductivefibrils allowed to adhere to the thread. The thread may then be twistedon itself or with a second or a plurality of similarly treated threads.Alternatively or in addition, the threads may be braided. The adhesivemay be a lipid that is waxy at room temperature, for example, a di- ortri-glyceride that is solid at room temperature. Alternatively or inaddition, the adhesive may be a phosphocholine or phosphatidylcholine.In some embodiments, the adhesive is a material that binds both thethread and the material that is used to coat the thread (e.g., boneparticles) but that does not degrade either. Non-aqueous adhesives mayimprove the stability of the final aggregate as compared to aqueousadhesives.

Suitable fibers may be formed utilizing well known techniques, e.g.,braiding, plying, knitting, weaving, felting, that are applied toprocessing natural fibers, e.g., cotton, silk, etc., and syntheticfibers made from synthetic bioabsorbable polymers, e.g., poly(glycolide)and poly(lactic acid), nylon, cellulose acetate, etc. Specifically,collagen thread is wound onto cylindrical stainless steel spools. Thespools are then mounted onto the braiding carousel, and the collagenthread is then assembled in accordance with the instructions providedwith the braiding machine. In one particular run, a braid was formed offour collagen threads, which consisted of two threads of noncrosslinkedcollagen and two threads of crosslinked collagen. One skilled in the artwill recognize that these techniques may be applied to the other fibrousmaterials described herein.

Fibers and more evenly dimensioned particles may also be plied intoyarns using the same methods and same machinery known to those skilledin the art in plying threads made out of other material, e.g., cotton,polyester, etc.

Elongated materials including multistranded materials, e.g., braids,plied yarns, cables, etc., may be knitted into tubular or flat fabricsby using techniques known to those skilled in the art of producingfabrics manufactured from other types of threads. Various biologicallyactive substances can be incorporated in, or associated with, thebraided, knitted, or woven materials. Particles and fibers and materialsof these (including multistranded materials) may alternatively oradditionally be assembled into a material by non-woven methods such aslaying, needle-punching, and hooking. For example, a thread may beattached to another thread or a pressed film.

Regardless of the assembly method, the material shape, mesh size, cablethickness, and other structural characteristics, e.g., architecture, maybe customized for the desired application. For example, where a twodimensional aggregate is used to retain a thixotropic material within agap, a tight weave is preferred to prevent leakage. To optimize cell orfluid migration through the mesh, the pore size may be optimized for theviscosity and surface tension of the fluid or the size of the cells. Forexample, pore sizes on the order of approximately 100-200 μm may be usedif cells are to migrate through the mesh. Mesh size may be controlled byphysically weaving strands of the material by controlling the ratio ofsolvent to solids in a precursor material.

Cells may be seeded onto the material, or contained within it. In oneembodiment, cells may be encapsulated in a matrix such as alginate orcollagen gel and the capsules placed on the material. Seeded materialsgenerally do not need to be incubated for long periods of time insolutions that could partially dissolve the binding agent. Instead, thecapsules may be placed on the material or covering shortly beforeimplantation. In another embodiment, cells are simply mixed with a gel,which is then combined with the material. Alternatively, a material orcovering may be cultured with cells before implantation. In oneembodiment, thicker materials are used for culturing to increasemechanical integrity during implantation. Any class of cells, includingconnective tissue cells, organ cells, muscle cells, nerve cells, andstem cells, may be seeded onto the implant. In an exemplary embodiment,connective tissue cells such as osteoblasts, osteoclasts, fibroblasts,tenocytes, chondrocytes, and ligament cells and partially differentiatedstem cells such as mesenchymal stem cells and bone marrow stromal cellsare employed.

Covering Configurations

The shape, configuration, or form of the covering may be selected forparticular applications. Such shape and configuration may include, forexample, the basic shape of the covering (e.g., a cylinder or a bag),whether the covering has a single or a plurality of compartments, andwhether the covering includes attachment mechanisms. The covering (ordelivery system) may be configured to conform to surrounding bonycontours of the space in which it is placed.

As previously discussed, the covering may be formed of as a mesh. Thus,the covering may comprise a woven material. The woven material may havevarying degrees of permeability. It may be permeable, semi-permeable, ornon-permeable. Permeability may be with respect to cells, to liquids, toproteins, to growth factors, to bone morphogenetic proteins, or other.In further embodiments, the material may be braided.

In alternative embodiments, the covering may comprise a substantiallysolid structure, such as a polymer structure with a chamber, or a spuncocoon.

The covering may have any suitable configuration. For example, thecovering may be formed as a ring, a cylinder, a cage, a rectangularshape, a mesh, a suture-like wrap, a continuous tube, or otherconfiguration. In specific embodiments, the covering may be formed as athin tube designed to be inserted through catheters or an introducertube, a rectangular shape designed to fit adjacent to spinal processesfor posterolateral spine fusion, a cube like structure designed to fitbetween vertebral bodies or within cages for interbody spinal fusion, atube-like shape where the ends are designed to be fitted onto nonunionlong bone defects, relatively flat shapes designed to fill cranial ormaxillofacial defects, rectangular structures designed for osteochondraldefects, structures preshaped to fit around various implants (e.g.,dental, doughnut with hole for dental implants), or relatively elasticring-like structures that will stretch and then conform to shapes (e.g.rubber band fitted around processes). In an embodiment wherein thecovering is formed as a cage, the cage may comprise a plurality ofcrossed filaments, which define between them a series of openings fortissue ingrowth. Any of these shapes may be used for a coveringcomprising a plurality of compartments. For example, in a tubularembodiment, the tube may be formed into a plurality of compartments bytying a cord around the tube at one or more points, or by other suitablemechanism such as crimping, twisting, knotting, stapling, sewing, orother method. The configuration of the covering may be determined by thesubstance to be provided within the covering. For example, if thesubstance to be contained comprises fibers, the covering may be formedas strings or sutures that are wrapped around the fibers.

In certain embodiments, a bone void can be filled. A compartment withinthe covering material can be at least partially filled with a bonerepair substance (e.g., demineralized allograft material). In variousembodiments, at least partially filled as used herein, can mean that apercentage of the volume of a compartment (or covering material, asapplicable) is at least 70% occupied, at least 75% occupied, at least80% occupied, at least 85% occupied, at least 90% occupied, at least 95%occupied, or 100% occupied. The covering material can be inserted intoan opening in the defect until the defect is substantially filled. Invarious embodiments, a substantially filled as used herein can mean thata percentage of the volume of a defect (or covering material, asapplicable) is at least 70% occupied, at least 75% occupied, at least80% occupied, at least 85% occupied, at least 90% occupied, at least 95%occupied, or 100% occupied. The excess material extending beyond thesurface of the bone if the bone were without the defect can then beremoved, or at least partially removed such that the opening of thedefect is flush with the uninjured bone surface.

A covering as provided herein may further comprise an attachment orcoupling mechanism. Any suitable attachment mechanism can be used, suchas a tab, loop, tack or other structure adapted for attachment at thesite. Also, for example, a covering may include a hook-and-eye (Velcro™)portion. The hook-and-eye portion may be used to couple the covering toa tissue structure, such as bone, or to another covering. For example, adual compartment covering may be formed by two single-compartmentcoverings coupled at complementary ends thereof. For example, thecoupling portion may comprise overlapping/mating Velcro™ portions. Thesize and shapes of the single compartment coverings may be the same ormay be different. Further, the materials of the compartment coveringsand the substances provided therein may be the same or may be different.The coupling may be done pre-implantation or post-implantation. Inpost-implantation embodiments, the coupling may be done by insertingfirst and second coverings through an opening into a space and couplingthe coverings within the space. Other suitable attachment, or coupling,mechanisms are described more fully below.

In some embodiments, the covering may be labeled. Such labeling may bedone in any suitable manner and at any suitable location on thecovering. In some embodiments, labeling may be done by using a silkscreen printing, using an altered weaving or knotting pattern, by usingdifferent colored threads, or other. The labeling may indicateinformation regarding the covering. Such information might include partnumber, donor id number, number, lettering or wording indicating orderof use in the procedure or implant size, etc.

Compartments

A covering may comprise a single compartment implant body in any shapeor form. In further exemplary embodiments, the covering may be a narrowtube for delivery through a catheter. For example, the covering may bedelivered percutaneously using a catheter through which it is inserted.Thus, the covering may have dimensions suitable for receipt in thecatheter. Optionally, the covering may be stiffened to facilitateinsertion into the catheter. Such stiffening may be achieved throughchoice of material for the covering, by treating the material of thecovering, or other. In some embodiments, the covering may be coated witha material to facilitate sliding engagement with the catheter.

As to volume, advantageous implant bodies can have a total volume of atleast about 2 cubic centimeters (cc), e.g. in the range of about 2 cc toabout 100 cc, and more typically in the range of about 10 cc to about 50cc, although both smaller and larger overall volumes may also be used.Similarly, the volume of the pieces into which the implant bodies areconfigured to be separated may range from about 1 cc to about 50 cc,more typically in the range of about 5 cc to about 20 cc, although otherpiece volumes will also be suitable in broader aspects of the presentprinciples.

In other examples, the covering of an implant body includes score linesfor enabling portions of the body to be separated. The delivery systemmay be used in its entirety at an implant site or may be manipulated bythe user to separate the implant body into multiple smaller pieces, someor all of which may be used, at a single implant site or at multipledifferent implant sites.

In one embodiment of a single compartment covering, a plurality ofsubstances may be provided within the covering based on characteristicsof the substances. For example, where it is desirable to include aparticulated first substance within a material having mesh openingslarger than the substance, a second substance may be providedsurrounding the particulated first substance (e.g., modifieddemineralized allograft material) to reduce the likelihood of release ofparticles of the first substance from the mesh. Thus, for example, aparticulated first substance and a particulated second substance may beprovided wherein the particles of the first substance have a smallersize than the particles of the second substance. A covering is providedcomprising a mesh having mesh openings or pores larger than theparticles of the first substance. For use, the first substance isprovided generally centrally within the covering, the second substanceis provided around the first substance and thus between the firstsubstance and the second substance. In further embodiments, the secondsubstance may be coated, for example via spray coating or solventcasting.

In yet a further embodiment, a single compartment covering may be usedas a spacer for nonunion. For example, the covering may be placed in acanal of a long bone.

Multi Compartment

In alternative embodiments, the covering may comprise a plurality ofcompartments. For example, the covering may comprise nested coverings,coverings coupled via a temporary barrier, coverings separated with aboundary, and others, described below. In embodiments comprising twocompartments, a second compartment may be adjacent, apart from, inside,or surrounding a first compartment. Materials for the first compartmentand the second compartment (which may be designated first and secondsubstances) may be the same, partially the same, or different. Thematerials for the first compartment and the second compartment may havedifferent release profiles, different porosities, and other differentcharacteristics. Selection of materials, positioning of thecompartments, and other factors relating to the first and secondcompartments may be chosen to achieve simultaneous or sequentialdelivery or release of a substance or substances. A first substance maybe provided in the first compartment and a second substance may beprovided in the second compartment. In some embodiments, anosteoinductive substance may be placed in a compartment generallyadjacent tissue being treated as implanted and an osteoconductivesubstance may be placed in a compartment not adjacent tissue beingtreated. Release rates for the materials provided in the firstcompartment and the second compartment may be different. In someembodiments, at least one of the compartments may be unfilled at thetime of surgery and autograft or other material may be provided thereinin the operating room or at the surgical site. In some embodiments, thecovering may form a 3D scaffold.

In any of the discussed examples the surgeon may select the number ofportions of the body or number of compartments desired for placement andcut or pull/tear along the score lines or perforations providing thedesired number of portions or compartments. Further, if the covering ismade of a material that is brittle or strain hardens, the surgeon mayseparate the compartments by bending the selected portion back uponitself and reversing until the segments separate. In some embodiments,every other compartment, for example, may be preloaded or filled with asubstance for delivery. Alternatively, only some of the compartments maybe preloaded, for example, every other compartment may be preloaded suchthat alternating compartments may be filled in the operating room or atthe surgical site.

In some embodiments, at least one but not all of the compartments may beweight-bearing. In other embodiments, all of the compartments may beweight-bearing.

In one embodiment, the covering may comprise a penetrable material at afirst compartment configured for placement adjacent bone and asubstantially impenetrable material at a second compartment configuredfor placement adjacent soft tissue. Alternatively, the material of thecompartments may have substantially identical characteristics. Thecovering then can be positioned in any desirable manner. By way ofexample only, a covering may have a porous surface that is positionedadjacent bone, and a separate or opposite surface that has a generallyimpenetrable surface that is positioned adjacent soft tissue.Alternatively, a covering may have one compartment that comprises aporous material, and a second compartment that comprises a substantiallyimpenetrable material.

In another embodiment, the covering may comprise a continuous tubewherein the tube may be twisted to divide portions of the tube. The tubethus may be divided into a series of implants, each having ends that maybe twisted or heat treated. Any suitable manner of dividing the tubeinto a plurality of compartments may be used. For example, the tube maybe crimped, heat treated, twisted, knotted, stapled, sewn, or otherwisedivided. Any suitable tool may be used for dividing the tube into suchcompartments including, for example, a crimper, a heat tool, or other.

In some embodiments, a multi-compartment covering may be configured tobe foldable and stackable.

For both single and multi-compartment coverings, the covering may beclosed after filling substances. Accordingly, the covering may beprovided in an unfilled, unsealed state. After a substance for deliveryis placed in the covering, the covering may be permanently ortemporarily closed. Permanent closure may be, for example, by heatsealing, stitching, adhesion, or other methods. Temporary closure may beby tying, fold lock, cinching, and etc. A temporarily closed coveringcan be opened without damaging the covering during surgical implantationto add or remove substances in the covering.

Attachment Mechanisms

The covering may be configured with structures to permit attachment atthe surgical site, such as to skeletal tissue or to soft tissuestructures, or for attachment to other coverings, or for attachment toadjacent implantable medical devices or products (such as a rod or screwor cross-brace of a pedicle screw fixation system, a hip prosthesis, abone plate, and the like). Generally, the attachment mechanism may beused to retain the covering at the surgical site and any mechanismscapable of doing so may be used. The attachment may be to bone or toadjacent tissues such as muscle, tendon, or ligament. Where the coveringretains a bone graft substance, the covering may be held in a relativelystable position relative to bone (or relative to the surgical site orsurgical defect) to promote bone growth. Accordingly, in someembodiments, the delivery system may be suitable for assisting inattaching tendons, artificial tendons, or ligaments to bone or otherstructure.

The bone or soft tissue to which the covering is attached may beprepared for receiving the attachment mechanism(s). For example, inspinal applications, slots or perforations may be provided in posteriorelements such as transverse processes, spinous processes, or other boneor tissue to receive the attachment mechanism.

Any suitable attachment mechanism may be used, including mechanical,physical, chemical, and biological attachment mechanisms. The attachmentmechanism may be provided at an end of the covering, centrally in or onthe covering, generally in or on the body of the covering, or anycombinations of these. When an attachment mechanism is used to couplefirst and second coverings to one another, such attachment or couplingmay be done pre-implantation or post-implantation. In post-implantationembodiments, the coupling may be done by inserting first and secondcoverings through an opening into a space and coupling the coveringswithin the space. In some embodiments, the covering may be provided withattachment mechanisms to facilitate suturing or other attachment of thecovering in vivo.

In some embodiments, a covering may include an area for receipt of anattachment mechanism. For example, a covering may include a tab forreceipt of a screw. In other embodiments, an attachment mechanism mayinterface with any portion of the covering. For example, a screwattachment mechanism may be threaded through a covering at any location,including central to a containment area of the covering. In someembodiments, a screw attachment mechanism may be threaded through thecovering and the substance provided in the containment area of thecovering.

A further method of attachment may comprise suturing or otherwiseattaching the covering to a tether, anchor, or screw embedded in a bonystructure, e.g. a pedicle screw of a spinal stabilization system. Suchscrew, anchor, or tether may pass through the covering and its containedcontents to provide fixation, or through a tab at a margin of thecovering, or through other structure of the covering.

Chemical attachment mechanisms may comprise, for example, a bioadhesiveor glue, cement, tape, tissue adhesives, or similar mechanism. Chemicalattachment mechanisms may further comprise mechanisms that facilitatecross-linking. In further embodiments, attachment mechanisms such ascrimping, welding, soldering, or brazing may be used. For example,tissue welding may be used. Further, attachment may be achieved viafriction.

Suitable adhesives for use may include, for example, cyanoacrylates(such as histoacryl, B Braun, which is n-Butyl-2 Cyanoacrylate; orDermabond, which is 2-octylcyanoacrylate); epoxy-based compounds, dentalresin sealants, dental resin cements, glass ionomer cements, polymethylmethacrylate, gelatin-resorcinol-formaldehyde glues, collagen-basedglues, inorganic bonding agents such as zinc phosphate, magnesiumphosphate or other phosphate-based cements, zinc carboxylate, L-DOPA(3,4-dihydroxy-L-phenylalanine), proteins, carbohydrates, glycoproteins,mucopolysaccharides, other polysaccharides, hydrogels, protein-basedbinders such as fibrin glues and mussel-derived adhesive proteins, andany other suitable substance. Adhesives may be selected for use based ontheir bonding time; e.g., in some circumstances, a temporary adhesivemay be desirable, e.g., for fixation during the surgical procedure andfor a limited time thereafter, while in other circumstances a permanentadhesive may be desired. Where the compartment is made of a materialthat is resorbable, the adhesive can be selected that would adhere forabout as long as the material is present in the body. In someembodiments, the covering material may be treated to form chemicallinkages between the covering and adjacent tissue, whether bone or softtissue.

In some embodiments, biological attachment may be via mechanisms thatpromote tissue ingrowth such as by a porous coating or ahydroxyapatite-tricalcium phosphate (HA/TCP) coating. Generally,hydroxyapatite bonds by biological effects of new tissue formation.Porous ingrowth surfaces, such as titanium alloy materials in a beadedcoating or tantalum porous metal or trabecular metal may be used andfacilitate attachment at least by encouraging bone to grow through theporous implant surface. These mechanisms may be referred to asbiological attachment mechanisms.

Generally, any combination of mechanical, physical, chemical, orbiological attachment mechanisms may be used.

Any of the various attachment mechanisms may be provided as part of thecovering or may be supplied separately. In various embodiments, theattachment mechanisms may be integral to the covering. Alternatively,the attachment mechanisms may be secured to the covering, for example,by stitching, welding, crimping, or other. The attachment mechanisms mayhave any suitable geometric configuration and may optionally includeapertures for receiving other components for coupling in vivo, such asan aperture for receiving a screw. Thus, for example, an attachmentmechanism may be provided configured for receiving an anchor forfixation to bone. Generally, any number of attachment mechanisms may beprovided at any suitable location on the covering.

The attachment mechanisms may be manufactured of the same material asthe portion of the covering to which it is coupled or may bemanufactured of a different material from the portion of the covering towhich it is coupled. The attachment mechanism may be resorbable ornonresorbable. The material of the attachment mechanism may be selectedto allow anchoring the covering to an adjacent covering having acomplementary attachment mechanism or to another structure. In variousembodiments, the attachment mechanism may comprise, allograft, syntheticmaterials, demineralized bone, nondemineralized bone, other material, orcombinations of these. The shape and size of the attachment mechanismmay be selected based on application.

In some embodiments, the covering may be tubular and have threaded endssuch that the ends may be threaded with a reciprocal thread of a furtherdevice or implant. For example, the covering may be used withinterference screws. In some embodiments, the covering may includeextensions or tabs that may be used for wrapping around or suturing tothe surgical site. Alternatively, the covering may be sutured directlyto the surgical site. The ends of the covering may be presealed or maybe sealed after introduction of contents. Sealing may be done by usingadhesives, heating, solvent treatment, suturing, knotting, or any othermeans.

Substance for Delivery by Covering

A substance may be provided in the covering, before or during surgery(as described herein), for delivery in vivo. Generally, the substance ormaterial may be homogenous or heterogeneous. The substance or materialmay be selected to exhibit certain gradients. For example, the substanceor material may be selected to exhibit a gradient to guide, lure, orattract cells along a pathway. Such gradient may comprise a cellgradient, a cell type gradient (for example transitioning from bonecells to cartilage cells or transitioning from bone cells to tendoncells), a gradient of conductivity, or a gradient of density/porosity.

The covering may be used to deliver a substance comprising any suitablebiocompatible material. In some embodiments, the substance may comprisea biocompatible material comprising a demineralized allograft bonematerial modified to provide an ionic gradient to produce a modifieddemineralized allograft bone material as per some embodiments of thepresent disclosure, which, when implanted, results in enhanced ingrowthof host bone. Namely, a demineralized allograft bone material may bemodified in such a way that the original chemical forces naturallypresent have been altered in such a way as to attract and bind growthfactors, other proteins and cells affecting osteogenesis,osteoconduction and osteoinduction.

In some embodiments, the demineralized allograft bone material comprisesdemineralized bone matrix fibers and demineralized bone matrix chips ina 30:60 ratio.

In some embodiments, the substance or material may comprise a sequenceof ingredients.

In specific embodiments, the covering may be used to deliver surfacedemineralized bone chips, optionally of a predetermined particle size,demineralized bone fibers, optionally pressed, and/or allograft. Forembodiments wherein the substance is biologic, the substance may beautogenic, allogenic, xenogenic, or transgenic. Other suitable materialsthat may be positioned in the covering include, for example, protein,nucleic acid, carbohydrate, lipids, collagen, allograft bone, autograftbone, cartilage stimulating substances, allograft cartilage, TCP,hydroxyapatite, calcium sulfate, polymer, nanofibrous polymers, growthfactors, carriers for growth factors, growth factor extracts of tissues,demineralized bone matrix, dentine, bone marrow aspirate, bone marrowaspirate combined with various osteoinductive or osteoconductivecarriers, concentrates of lipid derived or marrow derived adult stemcells, umbilical cord derived stem cells, adult or embryonic stem cellscombined with various osteoinductive or osteoconductive carriers,transfected cell lines, bone forming cells derived from periosteum,combinations of bone stimulating and cartilage stimulating materials,committed or partially committed cells from the osteogenic orchondrogenic lineage, or combinations of any of the above. In someembodiments, the substance may be pressed before placement in thecovering. A substance provided within the covering may be homogenous, orgenerally a single substance, or may be heterogeneous, or a mixture ofsubstances.

In some embodiments, the substance may be designed to expand in vivo.Such an embodiment may be used to fill a space and create contact withcongruent surfaces as it expands in vivo, for example for interbodyfusion. Thus, in some embodiments, the delivery system may be used inthe disc space, between implants, or inside a cage.

The covering retains the substance in place by pressure against thecovering. The covering thus may, in some embodiments, maintain particlesof substance in close proximity (for example, where the covering retainsa substance comprising bone particles). Generally, the ratio of coveringmaterial to substance for placement within the covering may be low. Forexample, in some embodiments, the ratio of covering material tosubstance, by weight, may be approximately 1:1,000, 1:100, 1:50, 1:25,1:1, or any suitable ratio that may be higher or lower than these.

In some embodiments the substance delivered by the covering may includeor comprise an additive such as an angiogenesis promoting material or abioactive agent. It will be appreciated that the amount of additive usedmay vary depending upon the type of additive, the specific activity ofthe particular additive preparation employed, and the intended use ofthe composition. The desired amount is readily determinable by oneskilled in the art. Angiogenesis may be an important contributing factorfor the replacement of new bone and cartilage tissues. In certainembodiments, angiogenesis is promoted so that blood vessels are formedat an implant site to allow efficient transport of oxygen and othernutrients and growth factors to the developing bone or cartilage tissue.Thus, angiogenesis promoting factors may be added to the substance toincrease angiogenesis. For example, class 3 semaphorins, e.g., SEMA3,controls vascular morphogenesis by inhibiting integrin function in thevascular system, and may be included in the recovered hydroxyapatite.

In accordance with some embodiments, the substance may be supplemented,further treated, or chemically modified with one or more bioactiveagents or bioactive compounds. Bioactive agent or bioactive compound, asused herein, refers to a compound or entity that alters, inhibits,activates, or otherwise affects biological or chemical events. Forexample, bioactive agents may include, but are not limited to,osteogenic or chondrogenic proteins or peptides; demineralized bonepowder; collagen, insoluble collagen derivatives, etc., and solublesolids and/or liquids dissolved therein; anti-AIDS substances;anti-cancer substances; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymycin B,tetracyclines, biomycin, chloromycetin, and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc;immunosuppressants; anti-viral substances such as substances effectiveagainst hepatitis; enzyme inhibitors; hormones; neurotoxins; opioids;hypnotics; anti-histamines; lubricants; tranquilizers; anti-convulsants;muscle relaxants and anti-Parkinson substances; anti-spasmodics andmuscle contractants including channel blockers; miotics andanti-cholinergics; anti-glaucoma compounds; anti-parasite and/oranti-protozoal compounds; modulators of cell-extracellular matrixinteractions including cell growth inhibitors and antiadhesionmolecules; vasodilating agents; inhibitors of DNA, RNA, or proteinsynthesis; anti-hypertensives; analgesics; anti-pyretics; steroidal andnon-steroidal anti-inflammatory agents; anti-angiogenic factors;angiogenic factors and polymeric carriers containing such factors;anti-secretory factors; anticoagulants and/or antithrombotic agents;local anesthetics; ophthalmics; prostaglandins; anti-depressants;anti-psychotic substances; anti-emetics; imaging agents;biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids;peptides; vitamins; inorganic elements; co-factors for proteinsynthesis; endocrine tissue or tissue fragments; synthesizers; enzymessuch as alkaline phosphatase, collagenase, peptidases, oxidases, etc.;polymer cell scaffolds with parenchymal cells; collagen lattices;antigenic agents; cytoskeletal agents; cartilage fragments; living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells; naturalextracts; genetically engineered living cells or otherwise modifiedliving cells; expanded or cultured cells; DNA delivered by plasmid,viral vectors, or other means; tissue transplants; autogenous tissuessuch as blood, serum, soft tissue, bone marrow, etc.; bioadhesives; bonemorphogenic proteins (BMPs); osteoinductive factor (IFO); fibronectin(FN); endothelial cell growth factor (ECGF); vascular endothelial growthfactor (VEGF); cementum attachment extracts (CAE); ketanserin; humangrowth hormone (HGH); animal growth hormones; epidermal growth factor(EGF); interleukins, e.g., interleukin-1 (IL-1), interleukin-2 (IL-2);human alpha thrombin; transforming growth factor (TGF-β); insulin-likegrowth factors (IGF-1, IGF-2); parathyroid hormone (PTH); plateletderived growth factors (PDGF); fibroblast growth factors (FGF, BFGF,etc.); periodontal ligament chemotactic factor (PDLGF); enamel matrixproteins; growth and differentiation factors (GDF); hedgehog family ofproteins; protein receptor molecules; small peptides derived from growthfactors above; bone promoters; cytokines; somatotropin; bone digesters;antitumor agents; cellular attractants and attachment agents;immuno-suppressants; permeation enhancers, e.g., fatty acid esters suchas laureate, myristate and stearate monoesters of polyethylene glycol,enamine derivatives, alpha-keto aldehydes, etc.; and nucleic acids.

In certain embodiments, the bioactive agent may be a drug. In someembodiments, the bioactive agent may be a growth factor, cytokine,extracellular matrix molecule, or a fragment or derivative thereof, forexample, a protein or peptide sequence such as RGD.

In one embodiment of a covering comprising two compartments, a firstgrowth factor may be provided for delivery by the first compartment anda second growth factor may be provided for delivery by the secondcompartment. The first and second growth factors may be provided withother substances. The first and second growth factors may be selected(and placed in respective compartment for positioning in vivo) based ondesired characteristics of the growth factor. For example, an angiogenicgrowth factor may be provided in the first compartment and anosteoinductive growth factor may be provided in the second compartment.

Similarly, the substance delivered by the first compartment and thesubstance delivered by the second compartment may be selected based ondesired characteristics of the compartment according to its placement invivo. Thus, for example, one compartment may have a substance that issubstantially osteoclast stimulating while another compartment may havea substance that is substantially osteoblast stimulating.

In one embodiment, demineralized bone fibers may be provided in thefirst compartment and surface demineralized bone chips may be providedin the second compartment. In this embodiment, the demineralized bonefibers may generally provide osteoinductive characteristics and thesurface demineralized chips may generally provide osteoinductive and/orosteoconductive characteristics. In use, the covering may be laid flaton the transverse process and positioned such that the firstcompartment, holding the demineralized bone fibers, is nearest thevertebral body and the second compartment, holding the surfacedemineralized bone chips, is farther from the vertebral body, or thecompartments may be positioned in any other desired configuration. Inanother embodiment, a covering may comprise first and secondcompartments wherein autograft may be placed in one of the compartmentsprior to placement of the covering in vivo, described more fully below.In other embodiments, three or more compartments may be used, asappropriate for the materials being delivered and the application of thecompartmented implant. More than one substance may be provided in acompartment. For example, surface demineralized bone chips anddemineralized bone fibers may be mixed and provided within a singlecompartment. Such mixture of substances within a single compartment maybe a substantially uniform mix or may be a plurality of substancesplaced in the compartment separately such that they are substantiallyunmixed. When multiple compartments are used, each compartment maycontain one or more substances. Exemplary substances that may beprovided in one or more compartments of the delivery system includecells from the osteogenic precursors, growth factors, angiogenic factorsand other active proteins including bone morphogenic proteins, andcellular scaffolding materials of natural or synthetic origin,antibiotics, and other substances described below.

In some embodiments, other medical devices may be provided within thecovering. For example, one or more electrical stimulator electrodes maybe provided within the covering.

Sterilization

The medical device including the covering and/or its contents may besterilizable. In various embodiments, one or more components of thecovering and/or its contents are sterilized by radiation in a terminalsterilization step in the final packaging. Terminal sterilization of aproduct provides greater assurance of sterility than from processes suchas an aseptic process, which require individual product components to besterilized separately and the final package assembled in a sterileenvironment.

In various embodiments, gamma radiation is used in the terminalsterilization step, which involves utilizing ionizing energy from gammarays that penetrates deeply in the device and/or covering. Gamma raysare highly effective in killing microorganisms, they leave no residuesnor have sufficient energy to impart radioactivity to the device. Gammarays can be employed when the device is in the package and gammasterilization does not require high pressures or vacuum conditions,thus, package seals and other components are not stressed. In addition,gamma radiation eliminates the need for permeable packaging materials.

In various embodiments, electron beam (e-beam) radiation may be used tosterilize one or more components of the device and/or covering. E-beamradiation comprises a form of ionizing energy, which is generallycharacterized by low penetration and high-dose rates. E-beam irradiationis similar to gamma processing in that it alters various chemical andmolecular bonds on contact, including the reproductive cells ofmicroorganisms. Beams produced for e-beam sterilization areconcentrated, highly-charged streams of electrons generated by theacceleration and conversion of electricity. E-beam sterilization may beused, for example, when the medical device and/or covering is includedin a gel.

Other methods may also be used to sterilize the device and/or coveringand/or one or more components of the device and/or covering, including,but not limited to, gas sterilization, such as, for example, withethylene oxide or steam sterilization.

Method of Use

The covering delivers the substance or substances in vivo. Such deliverymay be active, passive, by diffusion, or other. Active delivery mayinclude the degradation or decomposition of the covering with theinteraction of body fluids, extracellular matrix molecules, enzymes orcells. It may also include the cleavage of physical and/or chemicalinteractions of substance from covering with the presence of bodyfluids, extracellular matrix molecules, enzymes or cells. Further, itmay comprise formation change of substances (growth factors, proteins,polypeptides) by body fluids, extracellular matrix molecules, enzymes orcells.

The covering is loaded with the substance for placement in vivo. Thecovering may be pre-loaded, thus loaded at manufacture, or may be loadedin the operating room or at the surgical site. Preloading may be donewith any of the substances previously discussed including, for example,DBM, synthetic calcium phosphates, synthetic calcium sulfates, enhancedDBM, collagen, carrier for stem cells, and expanded cells (stem cells ortransgenic cells). Loading in the operating room or at the surgical sitemay be done with any of these materials and further with autograftand/or bone marrow aspirate.

Any suitable method may be used for loading a substance in the coveringin the operating room or at the surgical site. For example, thesubstance may be spooned into the covering, the substance may be placedin the covering using forceps, the substance may be loaded into thecovering using a syringe (with or without a needle), or the substancemay be inserted into the covering in any other suitable manner. Specificembodiments for loading at the surgical site include for vertebroplastyor for interbody space filler.

For placement, the substance or substances may be provided in thecovering and the covering placed in vivo. In one embodiment, thecovering is placed in vivo by placing the covering in a catheter ortubular inserter and delivering the covering with the catheter ortubular inserter. The covering, with a substance provided therein, maybe steerable such that it can be used with flexible introducerinstruments for, for example, minimally invasive spinal procedures. Forexample, the osteoimplant may be introduced down a tubular retractor orscope, during XLIF, TLIF, or other procedures. In other embodiments, thecovering (with or without substance loaded) may be placed in a cage, forexample for interbody fusion.

In continuous tube embodiments, the surgeon may divide the tube into thedesired number of compartments, using a crimper, heat tool or other.Alternatively, in an embodiment wherein the tube is perforated into aplurality of compartments, the surgeon may select the number ofcompartments desired and cut along the applicable perforation. In someembodiments, some of the compartments may be prefilled with a substancefor delivery and other compartments may be empty for filling by thesurgeon. For example, ever other compartment between perforations may bepreloaded or filled. The osteoimplant thus may be customized by fillingthe empty compartments with a desired substance.

For example, in some embodiments, a portion of the covering for example,one compartment of a multi-compartment covering, may be filled withautograft. Thus, the covering may be substantially empty prior tosurgery. During surgery, a surgeon may remove autograft from the patientand place the autograft in the substantially empty compartment. Suchplacement may be done in any suitable manner. In one embodiment, thecovering may be provided with a port for receiving an opening of aninjection device and the autograft may be injected into the covering.Alternatively, the autograft may be mixed with allograft, synthetics, orany other desired substances or combination of substances.

Attachment mechanisms provided on the covering may be used to couple thecovering to a site in vivo.

Applications

The covering may be used in any suitable application. In someembodiments, the covering may be used in healing vertebral compressionfractures, interbody fusion, minimally invasive procedures,posterolateral fusion, correction of adult or pediatric scoliosis,treating long bone defects, osteochondral defects, ridge augmentation(dental/craniomaxillofacial, e.g. edentulous patients), beneath traumaplates, tibial plateau defects, filling bone cysts, wound healing,around trauma, contouring (cosmetic/plastic/reconstructive surgery), andothers. The delivery system may be used in a minimally invasiveprocedure via placement through a small incision, via delivery through atube, or other. The size and shape may be designed with restrictions ondelivery conditions.

An exemplary application for using a delivery system as disclosed isfusion of the spine. In clinical use, the covering and deliveredsubstance may be used to bridge the gap between the transverse processesof adjacent or sequential vertebral bodies. The delivery system may beused to bridge two or more spinal motion segments. The coveringsurrounds the substance to be implanted, and contains the substance toprovide a focus for healing activity in the body.

In other applications, the delivery system may be applied to transverseprocesses or spinous processes of vertebrae.

Generally, the delivery system may be applied to a pre-existing defect,to a created channel, or to a modified defect. Thus, for example, achannel may be formed in a bone, or a pre-existing defect may be cut toform a channel, for receipt of the delivery system. The covering may beconfigured to match the channel or defect. In some embodiments, theconfiguration of the covering may be chosen to match the channel. Inother embodiments, the channel may be created, or the defect expanded oraltered, to reflect a configuration of the covering. The covering may beplaced in the defect or channel and, optionally, coupled usingattachment mechanisms.

At the time just prior to when the delivery system is to be placed in adefect site, optional materials, e.g., autograft bone marrow aspirate,autograft bone, preparations of selected autograft cells, autograftcells containing genes encoding bone promoting action, etc., can becombined with the covering and/or with a substance provided within thecovering. The osteoimplant can be implanted at the bone repair site, ifdesired, using any suitable affixation means, e.g., sutures, staples,bioadhesives, screws, pins, rivets, other fasteners and the like or itmay be retained in place by the closing of the soft tissues around it.

In accordance with various embodiments, a delivery system for deliveryof a substance in vivo is provided. The delivery system comprises acovering and a substance. The covering may be a single ormulti-compartment structure capable of at least partially retaining asubstance provided therein until the covering is placed at a surgicalsite. The covering may include separation-assist lines such asperforations or score lines. Upon placement, the substance may bereleased (actively or passively) to the surgical site. The covering mayparticipate in, control, or otherwise adjust, the release of thesubstance. The delivery system may be used to control availability of asubstances provided within the delivery system to cells and tissues of asurgical site over time.

Although the disclosure has been described with reference to someembodiments, persons skilled in the art will recognize that changes maybe made in form and detail without departing from the spirit and scopeof the disclosure.

1. A method for enhancing ingrowth of host bone comprising modifying atleast a portion of a demineralized allograft bone material to provide anionic gradient to produce a modified demineralized allograft bonematerial; placing the modified demineralized allograft bone materialinto a chamber disposed within a polymer mesh covering; and implantingthe modified demineralized allograft bone material.
 2. A method of claim1, further comprising contacting the modified demineralized allograftbone material with a molecule, a cell culture, or a combination thereof.3. A method of claim 1, wherein the demineralized allograft bonematerial and a fully mineralized bone material are disposed within thechamber.
 4. A method of claim 2, wherein the cell culture comprises atleast one of mesenchymal stem cells, periosteal cells, pluripotent stemcells, osteoprogenitor cells, osteoblasts, osteoclasts, bonemarrow-derived cell lines, or a combination thereof.
 5. A method ofclaim 1, wherein the modifying comprises an application of an ionicforce change agent.
 6. A method of claim 5, wherein the ionic forcechange agent comprises at least one enzyme, pressure, heat, sheer force,oxygen plasma, supercritical nitrogen or supercritical carbon,supercritical water or a combination thereof.
 7. A method of claim 1,wherein the polymer mesh covering is a bag comprising PGA or PLA, andthe demineralized allograft bone material disposed within the chambercomprises demineralized bone matrix fibers and demineralized bone matrixchips in a 30:60 ratio.
 8. A method of claim 1, wherein thedemineralized allograft bone material comprises a composite bone.
 9. Amethod of claim 8, wherein the composite bone comprises a bone powder, apolymer and demineralized bone particles.
 10. A method of claim 1,wherein the modifying comprises a one-to-one substitution of the calciumion in calcium hydroxyapatite with an element comprising at least one ofa lithium ion, sodium ion, potassium ion and cesium ion.
 11. A method ofenhancing the binding of growth factors and cell cultures to ademineralized allograft bone material, the method comprising applying aneffective quantity of an ionic force change agent to at least a portionof the surface of the demineralized allograft bone material to produce abinding-sensitized demineralized allograft bone material; implanting thebinding-sensitized demineralized allograft bone material into a hostbone; and administering to the binding-sensitized demineralizedallograft bone material a growth factor, a cell culture, or acombination thereof.
 12. A method of claim 11, wherein the growth factoror cell culture or a combination thereof is administered to thebinding-sensitized demineralized allograft bone material prior to theimplantation of the binding-sensitized demineralized allograft bonematerial into the host bone.
 13. A method of claim 11, wherein thegrowth factor, the cell culture, or the combination thereof is capableof binding to the binding-sensitized demineralized allograft bonematerial.
 14. A method of claim 11, wherein the ionic force change agentcomprises at least one of enzymes, pressure, heat, sheer force, oxygenplasma, supercritical nitrogen or supercritical carbon, supercriticalwater or a combination thereof.
 15. A method of claim 11, wherein thegrowth factor comprises at least one of BMP-2, rhBMP-2, BMP-4, rhBMP-4,BMP-6, rhBMP-6, BMP-7 (OP-1), rhBMP-7, GDF-5, LIM mineralizationprotein, platelet derived growth factor (PDGF), transforming growthfactor β (TGF-β), insulin-related growth factor-I (IGF-I),insulin-related growth factor-II (IGF-II), fibroblast growth factor(FGF), beta-2-microglobulin (BDGF II), rhGDF-5, or a combinationthereof.
 16. A method of claim 11, wherein the cell culture comprises atleast one of mesenchymal stems cells, periosteal cells, pluripotent stemcells, osteoprogenitor cells, osteoblasts, osteoclasts, a bonemarrow-derived cell lines, or a combination thereof.
 17. A method ofclaim 11, wherein the growth factor comprises tartrate-resistant acidphosphatase.
 18. A bone graft material comprising: a biocompatiblematerial comprising a demineralized allograft bone material, wherein atleast a portion of a demineralized allograft bone material is modifiedto provide an ionic gradient to produce a binding-sensitizeddemineralized allograft bone material.
 19. A bone graft material ofclaim 18, wherein the biocompatible material comprises demineralizedbone matrix fibers and demineralized bone matrix chips in a 30:60 ratio.20. A bone graft material of claim 18, wherein the demineralizedallograft bone material is modified using an ionic force change agentcomprising at least one of enzyme, pressure, heat, sheer force, oxygenplasma, or a combination thereof.